**Direct biophotolysis**

236 Biogas

The amount of electrons generated on absorption of organic compounds depends on the source of organic carbon. Even a slight difference in the molecular structure can lead towards completely different metabolic pathway. For example, D- and L-isomers of malate (after conversion into pyruvate) can easily join the TCA cycle. In this way the energy demand for hydrogen generation is met, whereas such a substrate as acetate is used in the other metabolic pathways: e.g. glyoxylate cycle, citramalate cycle, and ethylmalonyl-CoA pathway (Kars, 2010). The excess of electrons generated during assimilation of such substrates as glycerol or butyrate must be accepted during CO2 photoreduction. Therefore, when the only source of carbon is glycerol, it is not assimilated in significant amounts, which is changed after supplementation of glycerol with malate. Initially, malate is assimilated from the medium and evolution of CO2 occurs. In the second step of reaction,

Fig. 10. Simplified scheme of carbon metabolism in *Rhodobacter sphaeroides* bacteria (Kotay,

2008).

the evolved CO2 permits the use of glycerol as a substrate (Pike, 1975).

Cells of certain algae (eg. *Chlamydomonas reinhardtii, Chlorealla fusca*) or cyanobacteria are capable to split water into molecular hydrogen and oxygen under illumination.

$$\text{2H}\_2\text{O} + \text{light energy} \rightarrow 2\text{H}\_2 + \text{O}\_2\tag{14}$$

This process require absolutely anaerobic conditions. Light energy with wavelength lower than 680 nm is absorbed by photosystem II (PSII) and generate stream of electrons and protons originating from water. Other photosystem (PSI) is induced with light wavelength lower than 700 nm. This allows for transportation of electrons from PSII to PSI *via* chain of reductors called cytochrome *bf*. Electrons from PSI system are transferred *via* ferrodoxine to hydrogenase (algae or cyanobacteria) or nitrogenase (cyanobacteria) and these enzymes reduce protons to molecular hydrogen. In direct biophotolysis neither CO2 nor liqid metabolites are observed. Hydrogenase is very sensitive to oxygen and irreversibly inhibits its activity, therefore constant removal of oxygen is required (Das, 2008). Recent studies concentrate on elimination of sensitivity of algae towards oxygen (Benemann, 1997).

#### **Indirect biophotolysis**

This process can be performed with certain cyanobacteria (e.g. *Anabeana variabilis*) in two steps:

$$\text{12H}\_2\text{O} + 6\text{CO}\_2 + \text{light energy} \rightarrow \text{C}\_6\text{H}\_{12}\text{O}\_6 + 6\text{O}\_2\tag{15}$$

$$\text{C}\_6\text{H}\_{12}\text{O}\_6 + 12\text{H}\_2\text{O} + \text{light energy} \rightarrow 12\text{H}\_2 + 6\text{CO}\_2\tag{16}$$

Different periods of oxygen and hydrogen generation allows to eliminate the inhibiting effect of oxygen on enzymes. Similarly, as in direct biophotolysis, photons activate PSI and PSII. In the presence of RuBisCO enzyme the CO2 adsorption occurs, what in consequence of photosynthetic reactions generate glucose and oxygen . In the second step in presence of hydrogense and nitrogenase (Kars, 2009) the decomposition of organic compound occurs.

This type of metabolism in industry is difficult to perform because of periodicity of process. The hydrogen yields generated either by direct or indirect photolysis are unfortunately very low in comparison with other fermentative methods (Das, 2008).

Microbiological Methods of Hydrogen Generation 239

Organic substrate is oxidized to CO2 in the cycle of tricarboxylic acids (TCA). Generated in this process electrons are transferred to nitrogenase *via* many carriers (e.g. NAD and ferredoxin). Nitrogenase reduce protons to molecular hydrogen. The photosynthetic apparatus acts simultaneously with TCA cycle transforming light into chemical energy. Here, the ATP with protons and electrons are directed towards nitrogenase. Photosynthetic bacteria contain the reverse hydrogenase enzyme which oxidize hydrogen back to protons. The final amount of photogenerated hydrogen is the difference between hydrogen formed in presence of nitrogenase and hydrogen consumed by reversed hydrogenase. The main advantage of this process rely on the high yield of hydrogen while transforming organic

Dark fermentation can occur in the absence of light. Anaerobic microorganisms are generating hydrogen while transforming biodegradable substances under oxygen free conditions. Unfortunately hydrogen is not the only gaseous product of this process. Carbon dioxide, methane, hydrogen sulfide can be found among generated gases, as well as liquid metabolites such as simple volatile fatty acids (VFA) and simple alcohols. In the presence of hydrogenase an organic compound is transformed in glycolysis (17) process into pyruvate. Next, it is oxidized to acetylo-Co-A with the reduction of ferredoxin (18). In the third step ferrodoxine is oxidized and evolved electrons are directed to protons and formation of

Glucose → pyruvate (17)

Pyruvate + CoA + 2Fd(ox) → acetylo-C-A + 2 Fd(red) + CO2 (18)

2Fd (red) → 2Fd(ox) + H2 (19) Theoretically, one mole of glucose should generate 4 moles of hydrogen and acetic acid in dark fermentation process. In practice, this yield is lower (2.5 – 2.7 moles of H2). Final amount of generated hydrogen depends on many factors including type and concentration of the substrate, pH value, hydraulic time of retention, substrate to inoculum ratio, Fe ions concentration etc. The relatively high rate of hydrogen production is the important factor

Application of hybrid systems allows the use of apparently useless and difficult to operate substrates in the photofermentation process. These compounds (e.g. saccharides) are decomposed in dark fermentation process into simple organic acids (e.g. acetic or butyric) which further undergo photofermentation by PNS bacteria. In one-step hybrid systems both types of bacteria grow in one pot. The amount of generated hydrogen comes from two

Synchronization of activity of both types of bacteria cultures is the main parameter in hybrid system. The rate of reaction is usually much higher in dark fermentation than in photofermentation. As a result of this discrepancy, the excessive accumulation of VFA and

compounds to H2 and CO2.

molecular hydrogen (19).

**Hybrid systems** 

*One-step hybrid system* 

influencing possible industrial applications.

processes occurring almost simultaneously.

**Dark fermentation** 

Fig. 11. Scheme of indirect photolysis of water (Maness, 2001)

#### **Photofermentation**

This process is based on decomposition of organic compounds to hydrogen in the absence of both oxygen and nitrogen but in presence of photosynthetic bacteria under illumination. Scheme on Fig. 12 shows the process of photofermentation catalyzed by nitrogenase.

Fig. 12. Scheme of hydrogen generation in photofermentation process (Koku, 2002)

Organic substrate is oxidized to CO2 in the cycle of tricarboxylic acids (TCA). Generated in this process electrons are transferred to nitrogenase *via* many carriers (e.g. NAD and ferredoxin). Nitrogenase reduce protons to molecular hydrogen. The photosynthetic apparatus acts simultaneously with TCA cycle transforming light into chemical energy. Here, the ATP with protons and electrons are directed towards nitrogenase. Photosynthetic bacteria contain the reverse hydrogenase enzyme which oxidize hydrogen back to protons. The final amount of photogenerated hydrogen is the difference between hydrogen formed in presence of nitrogenase and hydrogen consumed by reversed hydrogenase. The main advantage of this process rely on the high yield of hydrogen while transforming organic compounds to H2 and CO2.

#### **Dark fermentation**

238 Biogas

This process is based on decomposition of organic compounds to hydrogen in the absence of both oxygen and nitrogen but in presence of photosynthetic bacteria under illumination.

Scheme on Fig. 12 shows the process of photofermentation catalyzed by nitrogenase.

Fig. 12. Scheme of hydrogen generation in photofermentation process (Koku, 2002)

Fig. 11. Scheme of indirect photolysis of water (Maness, 2001)

**Photofermentation** 

Dark fermentation can occur in the absence of light. Anaerobic microorganisms are generating hydrogen while transforming biodegradable substances under oxygen free conditions. Unfortunately hydrogen is not the only gaseous product of this process. Carbon dioxide, methane, hydrogen sulfide can be found among generated gases, as well as liquid metabolites such as simple volatile fatty acids (VFA) and simple alcohols. In the presence of hydrogenase an organic compound is transformed in glycolysis (17) process into pyruvate. Next, it is oxidized to acetylo-Co-A with the reduction of ferredoxin (18). In the third step ferrodoxine is oxidized and evolved electrons are directed to protons and formation of molecular hydrogen (19).

$$\text{Glucose} \to \text{pyruvate} \tag{17}$$

$$\text{Pyruvate} + \text{CoA} + 2\text{Fd(ox)} \rightarrow \text{acetyl-C-A} + 2\text{ Fd(red)} + \text{CO}\_2 \tag{18}$$

$$\text{2Fd (red)} \rightarrow \text{2Fd(ox)} + \text{H}\_2 \tag{19}$$

Theoretically, one mole of glucose should generate 4 moles of hydrogen and acetic acid in dark fermentation process. In practice, this yield is lower (2.5 – 2.7 moles of H2). Final amount of generated hydrogen depends on many factors including type and concentration of the substrate, pH value, hydraulic time of retention, substrate to inoculum ratio, Fe ions concentration etc. The relatively high rate of hydrogen production is the important factor influencing possible industrial applications.

#### **Hybrid systems**

#### *One-step hybrid system*

Application of hybrid systems allows the use of apparently useless and difficult to operate substrates in the photofermentation process. These compounds (e.g. saccharides) are decomposed in dark fermentation process into simple organic acids (e.g. acetic or butyric) which further undergo photofermentation by PNS bacteria. In one-step hybrid systems both types of bacteria grow in one pot. The amount of generated hydrogen comes from two processes occurring almost simultaneously.

Synchronization of activity of both types of bacteria cultures is the main parameter in hybrid system. The rate of reaction is usually much higher in dark fermentation than in photofermentation. As a result of this discrepancy, the excessive accumulation of VFA and

Microbiological Methods of Hydrogen Generation 241

decomposition of formic acid in presence of formate-hydrogen liaze (FHL) representing the set of enzymes localized in the inner cell membrane. Hydrogenase 3 coded as *hycA* and formate dehydrogenase known as *fdhF* are the main components of the FHL. The presence of *hycA* gene limits the synthesis of *fhlA*, responsible for better activity of FHL towards hydrogen. Therefore the removal of hycA increases the *fhlA* gene expression and in consequence hydrogen production by 5-10%. (Hallenback, 2009). The research of the FHL genes expression were performed by Bisaillon *et al*. and other authors (Bisaillon, 2006, Turcot, 2008, Penfold, 2003) and they found almost two times higher rate of hydrogen generation for modified strain of E. coli HD701. Genes responsible for nickel-iron hydrogenases (hydrogenase I and II) coded by *hya* and *hyb* operons were found in the *E. coli* genom as well. It was found that elimination of these enzymes by genetic modification can result with almost 35% higher production of hydrogen (Hallenback, 2009, Bisaillon, 2006, Turcot, 2008). Other profits originating from genetic engineering are related to deactivation of enzymes responsible for transformations of glucose into lactic, succinic and fumaric acids. The removal of *ldhA* (lactic acid) and *frdBC* (succinic and fumaric acids) genes results in increase of hydrogen formation. The 1.4 fold higher amount of hydrogen were found by Yoshida *et al.* (Yoshida, 2006) in this situation. The new mutant strain of SR 15 can produce 1.82 mol H2/mol glucose what is close to the theoretical value (2 mol H2/mol glucose). Studies performed by *Maeda et al.* (Maeda, 2007) showed that bacteria BW2513 with seven modified genes (*hyaB, hybC, hycA,fdoG, frdC*, *ldhA and aceE*) generate 4.6 fold more hydrogen

The nitrogenase and uptake hydrogenase play an important role in the photofermentation process of hydrogen generation by PNS bacteria. The engineering of the mutants free of uptake hydrogenase is the basic task of gene modifications. Genes coding hydrogenase (*hup*) can be modified by resistance gene insertion into the *hup* genes or by deletion of *hup* genes (Kars, 2009, Kars, 2008, Kim, 2006). Appropriately modified *Rhodobacter spheroids* can

Production of polyhydroxybutyrate (PHB) accompany hydrogen generation by PNS bacteria what applies the excess of reducing equivalents in other metabolic pathway. The PHB is the storage material stored in cytoplasm. This compound is formed in the environment rich in carbon compounds but lean in nitrogen (Kemavongse, 2007). The PHB is unwanted competition product accompanying hydrogen generation. The removal of genes responsible for formation of PHB syntase effectively stops generation of the polymer (Kim, 2006). Low activity in PHB formation not always results in an increase of hydrogen yield. Whereas in presence of lactate, malate or malate the amount of photogenerated hydrogen is not influenced by PHB (Hustede, 1993) the presence of acetate can increase photofermentation towards hydrogen. However, the importance of PHB as biodegradable polymer significantly increased in recent years. Therefore, simultaneous photogeneration of hydrogen and PHB

There are genetic modifications influencing changes in the amount of LHC (light harvesting complexes). The reduction of pigment present in LHC diminish the self-shadow effect and therefore better access of light into deeper located cells. The decrease of amount of LH1 (Vasilyeva, 1999) complexes with maximum of absorption at 875 nm or those with absorption maximum at 800 and 850 nm (LH2) (Kim, 2006) can increase the amount of photo generated hydrogen. Genetic manipulations cannot lead to total elimination of the pigments (Kim, 2006).

generate hydrogen also in the absence of light (Kim, 2008).

gained economic dimension (Yigit, 1999).

than wild-type strain.

alcohols is observed. High concentration of VFA in the medium leads to the substrate inhibition (Kargi, 2010) as well as to the lowering of pH value (Liu, 2010) which consequently decreases the hydrogen yield or completely stops hydrogen generation. In hybrid systems photofermentation is the rate limiting step and slows down the overall reaction rate (Ozmihci, 2010). The unfavorable effect caused by the difference in the reactions rates can be counteracted by appropriate choice of concentrations of different strains of bacteria. The optimum concentration ratios can vary from 1:3.9 (Argun, 2010) even to 1:600 (Liu, 2010) depending on the strains of bacteria and types of substrates. The use of hardly soluble substrates such as e.g. starch leads towards formation of suspensions and flocculation of bacteria cells and further to limited accessibility of organic carbon to the bacteria and decrease in the yield of microbiologically generated hydrogen (Argun, 2009).

The main advantage of one-step hybrid systems is the high rate and much higher yield of hydrogen produced in comparison to those obtained in the process of dark fermentation performed by one culture only. Further increase in the yield of hydrogen generated by hybrid systems can be achieved by application of two-step systems (Argun, Kapdan 2009).

#### *Two-steps hybrid systems*

The yield of hydrogen generation in the photofermentation process can be lowered by low access of light, inappropriate concentration of the medium, substrate inhibition, presence of ammonium ions or other contaminants (Ozmihci, 2010). Because a much greater number of parameters influence the yield of hydrogen in photofermentation than in dark fermentation, the former process should be performed in an independent photobioreactor. Application of two-step hybrid systems allows the use of wastes containing inhibitors of photofermentation process (e. g. ammonium ions) (Azbar, 2010). These inhibitors are neutral for bacteria engaged in the dark fermentation.

Moreover, separation of these processes into two-step hybrid systems extends the list of organic substrates as it permits the use of highly thermophilic bacteria operating in temperatures higher than 70 °C (Ozgur, 2010). The natural organic substrates and wastes that can be used in two-step hybrid system. One mole of glucose theoretically generates four moles of hydrogen in dark fermentation, whereas acetic acid is the only side product (Antgenent, 2004). In practice, dark fermentation of liquid wastes generates much lower amounts of hydrogen (2.5-2.7 mole H2 per mole of glucose in waste) (Ueno, 1998, Yokoi, 2001, Yokoi, 2002). The hybrid systems are much more efficient. These results suggest that further development of two-step hybrid system can lead towards effective, economically feasible commercial applications.
